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European Journal of Biotechnology and Bioscience 2014; 1 (5): 07-13 ISSN 2321-9122 EJBB 2014; 1 (5): 07-13 Received 14-05-2014 Accepted: 18-05-2014 Kalyani A.L.T Research Scholar, AU College of Pharmaceutical Sciences, Pharmaceutical Biotechnology Division, Andhra University, Visakhapatnam 530003, India Naga Sireesha G AU College of Pharmaceutical Sciences, Pharmaceutical Biotechnology Division, Andhra University, Visakhapatnam 530003, India. Aditya A.K.G AU College of Pharmaceutical Sciences, Pharmaceutical Biotechnology Division, Andhra University, Visakhapatnam 530003, India. Girija Sankar G AU College of Pharmaceutical Sciences, Pharmaceutical Biotechnology Division, Andhra University, Visakhapatnam 530003, India. Prabhakar T AU College of Pharmaceutical Sciences, Pharmaceutical Biotechnology Division, Andhra University, Visakhapatnam 530003, India. Correspondence: Kalyani, Allada Lakshmi Tripura AU College of Pharmaceutical Sciences, Pharmaceutical Biotechnology Division, Andhra University, Visakhapatnam 530003, India. Email: [email protected] Tel: +91 94405 92934 Fax: 0891-2755547

Production Optimization of Rhamnolipid Biosurfactant

by Streptomyces coelicoflavus (NBRC 15399T) using Plackett-Burman design

Kalyani A.L.T, Naga Sireesha G, Aditya A.K.G, Girija Sankar G, Prabhakar T ABSTRACT Surfactants are amphipathic molecules which reduce surface tension, are widely used in pharmaceutical, cosmetic and food industries. In the present study, Streptomyces coelicoflavus, the biosurfactant producing actinomycetes, was isolated frogm soil contaminated with petroleum oil near the naval dockyard, Visakhapatnam. This isolate was then grown in Kim’s medium for extracellular biosurfactant production, and was evaluated by measuring emulsification index, surface tension, lipase production and para film-M method. Further quantification of Rhamnolipid biosurfactant was done by orcinol assay. The Plackett-Burman design was adopted to evaluate the amount of biosurfactant produced. Among the seven variables of Kim’s medium, three variables, namely, concentration of olive oil, NaNO3 and level of inoculums, were identified to cause a significant effect on the biosurfactant production. When the strain Streptomyces coelicoflavus was cultivated in the optimized medium, the biosurfactant production was increased to 475.24 µg/ml compared to un-optimized medium which was 458.78 µg/ml.

Keywords: Streptomyces coelicoflavus, Biosurfactant, Orcinol assay, Optimization, Plackett-Burman.

1. Introduction However, production of the biosurfactants in large quantities is the primary constraint for their industrial applications. In this context, development of a production process that ensures the biosurfactant production in large quantities, gains high importance. To do this, observing and understanding the microorganism and its response to a set of measurable environmental conditions becomes essential to develop an assured production process [12]. As part of this, development of a suitable culture medium, in which, the microorganism sustains and enhances its rate of metabolism is the primary task. The next task is the identification of suitable nutrients and their supply dosage to enhance the biosurfactant production capacity of the microorganism. In this context, the present study deals with optimizing the production medium culture by using Plackett-Burman experimental design to maximize the biosurfactant production by Streptomyces coelicoflavus. 2. Materials and methods 2.1 Sample collection from oil contaminated soil The soil sample was collected in sterile plastic bags from the naval dockyard in Visakhapatnam. Soil mixed with petrochemicals was taken for screening of biosurfactant producing actinomycetes. 2.2 Isolation of biosurfactant producing actinomycetes Collected soil sample was air dried at room temperature for 1 week, then was treated at 55 0C in a hot air oven for 3 h, and then was stored at room temperature in sterile bags. The sample was labelled as NDYS. About one gram of soil sample was weighed and transferred to 50 ml of sterile water, and then was placed on a rotary shaker at 120 rpm for 30 min. Then, serial dilutions were made from these solutions up to 10-10 order by taking 1 ml and diluting it to 10 ml with distilled water.

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European Journal of Biotechnology and Bioscience

Humic acid supplemented actinomycetes selective medium was used for specially obtaining biosurfactant producing actinomycetes [13]. 100 µl aliquots of the appropriate dilution were applied to Humic acid-Salts-Vitamin agar plates which the pH was adjusted to 7.0. These plates were supplemented with Rifampicin (50 µg/ml) and cycloheximide (50 µg/ml) and were incubated at 28 0C for 7 days for the growth of actinomycetes colonies. Humic acid-salts-vitamin agar medium was used to isolate specifically the biosurfactant producing actinomycetes [14]. After 7 days of incubation, actinomycetes colonies were preliminarily selected based on colony morphology and small portion of colony streaked on Bennets agar medium with the help of a sterile inoculating loop. 2.3 Semi-qualitative evidence of biosurfactant production Isolates were sub-cultured from the working stocks, and were incubated at 28 0C for 7 days. After incubation, the microbial growth from each slant was suspended in 2 ml of sterile distilled water, and was transferred into 50 ml of Kim’s medium in a 250 ml Erlenmeyer flask [15]. This flask was then incubated on a rotary shaker for 7 days at 28 0C. After incubation, the content of the flask was centrifuged at 7000 rpm for 20 min, and was filtered. This cell-free supernatant was used for experimentation in the following preliminary tests for identifying the presence of biosurfactant. 2.3.1 Lipase production: Tributyrin agar medium (Yeast extract 0.4%, Malt extract 1%, Dextrose 0.4%, Agar 2%, Tributyrin 1%) was prepared and inoculated with isolated pure cultures of NDYS isolates. After incubation of 7 days at 28 oC, clear zone around the organism indicates the production of lipase, which is the characteristic feature of biosurfactant producing organisms [16]. 2.3.2 Emulsification index: Emulsifying capacity of the biosurfactant was evaluated by emulsification index (E24). The E24 of the culture sample was determined by adding 6ml of kerosene, 4 ml of water and 1 ml of the cell-free broth in a test tube, and was vortexed at high speed for 2 min, and allowed to stand for 24 h. The E24 index is given as percentage of the height of the emulsified layer (cm) divided by the total height of the liquid column (cm). The percentage of emulsification index was calculated by using the following equation [17, 18].

2.3.3 Surface tension Measurement: Surface tension of the liquid was measured with a Stalagmometer by Drop count method [19]. Surface tension was determined on the basis of number of drops which fall per volume, the density of the sample and the surface tension of the reference liquid (water).

Where, σL is the surface tension of the liquid under investigation, σW is the surface tension of water, NL is the

number of drops of the liquid, NW is the number of drops of water, ρL is the density of the liquid, and ρW is the density of water. 2.3.4 Para film-M test: One drop of Bromophenol blue indicator was added to 2 ml of cell-free supernatant. 10 µl of this sample was carefully placed like a drop on Para film-M with a micropipette. The shape of this drop on the surface was inspected after 1 min. Sodium lauryl sulfate and distilled water were used as positive and negative controls respectively. If the drop becomes flat, it indicates the presence of biosurfactant. If it remains in a dome shape, it indicates the absence of biosurfactant [20]. 2.4 Quantification of Rhamnolipids 400 µL of cell free supernatant was taken and its pH was adjusted to 2 by adding 2N HCl to separate the Rhamnolipid. To this, 750 µl of diethyl ether was added and mixed thoroughly to extract Rhamnolipid into an organic layer. This organic layer was collected. Solvent addition and extraction were repeated twice. Ether fractions were pooled and dried by evaporation. 400 µl of pH 8 phosphate buffer was added to the remaining precipitate (this equals to 1x dilution). 2.7 ml of orcinol was added to 300 µl of the sample. 300 µl of phosphate buffer, taken as blank, was also treated with 2.7 ml of orcinol reagent. Standard solutions of L-Rhamnose between 50-250 µg/ml were prepared with phosphate buffer, and were treated with orcinol reagent. Samples, standards and blank were boiled for 20 min, and test solutions were left in darkness for 35 minutes to cool down to room temperature. Then, optical density was measured at 421 nm. The Rhamnolipid concentrations were calculated from standard graph prepared with L-rhamnose and were expressed as rhamnose equivalents [21, 22]. 2.5 Optimization of biosurfactant production by Plackett-Burman design An 8-Run Plackett- Burman design was applied to reflect the relative importance of various fermentation factors involved in the production of biosurfactant by NDYS-4 [23]. For each variable a high (+) and low (-) levels were tested. The examined variables in this experiment and their levels are shown in Table 1. Eight different trials were performed in duplicates. The main effect of each variable was determined by the following equation:

Exi = (Mi+ – Mi-) / N Where, Exi is the variable main effect, and Mi+, Mi- are the emulsification activity in the trials, where the independent variable was present in high and low concentrations, respectively, and N is the number of trials divided by 2. The statistical t-value for equal unpaired samples was calculated using STATISTICA-7 to determine the variable significance [24]. This design was applied with nine different fermentation conditions as shown in Table 2 (9th row represents the basal control). All experiments were performed in duplicates and the averages of the results of Orcinol assay were taken. The data from the experiment was used to calculate the effects and to determine the statistical significance of those effects. The variables with p-value < 0.05 were considered to have a

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significant effect on biosurfactant production

Table 1: Independent variables affecting surfactant Production and their levels in the Plackett-Burman experiment

Factor (g/l) Symbol Level +1 0 -1

Olive oil (ml) OO 32 30 28 NaNO3 Na 1.2 1 0.8 KH2PO4 KH 0.12 0.1 0.08

MgSO4.7H2O Mg 0.12 0.1 0.08 CaCl2 Ca 0.12 0.1 0.08

Yeast extract Ye 0.22 0.2 0.18 Level of inoculum (ml) Li 102 100 98

3. Results and Discussion 3.1 Isolation and Screening of Biosurfactant Producing Actinomycetes A total of 10 strains of actinomycetes were isolated from oil contaminated naval dockyard soil named NDYS. These strains were initially screened for Lipase production, which is the characteristic feature of biosurfactant producing actinomycetes [16]. Out of these 10 strains, three strains, namely, NDYS-1, NDYS-3, NDYS-4, had shown lipase

production, as shown in Fig.1. Further, these isolates were screened for extracellular biosurfactant production grown on Kim’s medium containing olive oil as the sole source of carbon [15]. These three isolates were then tested for biosurfactant production by parafilm-M test, where a flat drop was shown by NDYS-4 taking sodium laryl sulphate as positive control and distilled water as a negative control [20]

, as shown in Fig. 2.

Fig 1: Lipase Production for (NDYS-4)

Fig 2: Para film M- Method for NDYS Isolates

3.2 Evaluation of biosurfactant activity

In this study, biosurfactant activity was evaluated by measuring the surface tension by stalagmometer and the emulsification index [18, 19]. These tests were performed with

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cell-free supernatant of the isolates NDYS-1, NDYS-3 and NDYS-4, un-inoculated medium (control), and 10% sodium lauryl sulphate (SLS), taken as standard [26]. Out of three isolates, only NDYS-4 had shown highest of emulsion in 72 h and the surface tension measured was 27.96 dynes/cm, which was very close to the surface tension of the standard sodium lauryl sulphate. These results are shown in Fig. 3. From the above results, it was concluded that the isolate NDYS-4 was a potential bio-surfactant producer. 3.3 Conformation assay for Rhamnolipid In the present study, Orcinol assay was performed for the conformation of Rhamnolipid. In this assay, only NDYS-4 isolate had shown Rhamnolipid production [21]. This had confirmed that the isolate NDYS-4 is a potential producer of

glycolipids. The types of glycolipid were found to be rhamnolipid in nature. Therefore, this isolates NDYS-4 was selected for quantification of rhamnolipid by taking L-Rhamnose as standard. 3.4 Time course of biosurfactant production The biosurfactant production was dependent on growth of culture in the fermentation medium at about 3rd day of growth, surfactant concentration started to increase, reaching its maximum after about 5th day, there was a decrease in surfactant concentration. This indicated that the biosurfactant production had occurred predominantly in the exponential growth phase, the results are shown in Fig. 4 [27].

Fig 3: Evaluation of Biosurfactant Activity

Fig 4: Every day Biosurfactant Production Assay for NDYS-4 Isolate 3.5 Identification of selected isolate The selected isolate NDYS-4 was identified as Streptomyces coelicoflavus NRBC 15399T by IMTECH Chandigarh, India by analysing the phylogenetic tree. 3.6 Optimization of biosurfactant production by Plackett-Burman design Plackett-Burman design is one of the screening designs used for identifying important factors from among many potential factors. In this, usually only main effects are estimated. In

the traditional method, screening for each category of the sources is done at an arbitrarily selected level of each source, one category at a time, while keeping the other ingredients constant, again at arbitrarily selected levels. The difficulty is also faced in selecting the levels at which to fix the ingredients of other categories and also the selection of their sources. But in the Placket-Burman design, the data generated are used to select a few compounds in each category, based on highest product promotion. Different levels of the selected compounds are then evaluated to

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achieve optimum level. The interactive effects among the sources of different categories are ignored completely. So, statistical experimental designs are powerful tools for searching the key factors rapidly from a multivariable system

and minimizing the error in determining the effect of the ingredients. Therefore, results are achieved in an economical manner [28, 29].

Table 2: Results of the Plackett-Burman experimental design for 7 factors

Runs OO Na Kh Mg Ca Ye Level of inoculum

Biosurfactant concentration (µg/ml)

1 +1 -1 -1 -1 -1 +1 +1 464.59 2 -1 -1 -1 +1 +1 +1 -1 437.95 3 -1 -1 +1 +1 -1 -1 +1 448.61 4 -1 +1 -1 -1 +1 -1 +1 459.74 5 +1 +1 +1 +1 +1 +1 +1 475.24 6 -1 +1 +1 -1 -1 +1 -1 462.65 7 +1 +1 -1 +1 -1 -1 -1 467.98 8 +1 -1 +1 -1 +1 -1 -1 447.64 9 0 0 0 0 0 0 0 458.78

In this study, Plackett-Burman design was employed to evaluate the significant effect on seven different culture elements on the production of biosurfactant by Streptomyces coelicoflavus NRBC 15399T using a basal medium. The main effect, t-values and p-values were estimated for each independent variable on biosurfactant production as shown in Table 3 and graphically represented in Fig.5. The results indicated that the presence of high levels of olive oil,

NaNO3, KH2PO4, Yeast extract, level of inoculum in the growth medium affects biosurfactant production positively. On the other hand, the presence of MgSO4, CaCl2 at their lowest levels would result in high biosurfactant production. The same was confirmed from the Pareto graph shown in Fig.6, which indicates higher effects were presented in the upper portion and then progress down to the lower effects.

Table 3: Statistical analysis of effects of medium constituents on biosurfactant production as per PBD

Variables Medium components (g/l) Effect Standard error t-value p- value X1 Olive oil (ml) 11.62 0.486 23.88 0.026 X2 NaNO3 16.70 0.486 34.32 0.018 X3 KH2PO4 0.97 0.486 1.99 0.29 X4 MgSO4.7H2O -1.21 0.486 -2.48 0.24 X5 CaCl2 -5.81 0.486 -11.94 0.05 X6 Yeast extract 4.11 0.486 8.45 0.07 X7 Level of inoculums (ml) 7.99 0.486 16.41 0.03

*t-value significant at 1% level = 3.70; 5% level = 2.45; 10% level = 1.94; 20% level = 1.37. Standard t-values obtained from statistical methods (Cochran and Snedecor, 1989)

Fig 5: Pareto chart showing the effect of different media components (variables) on biosurfactant production based on the

observations of Plackett-Burman design

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On the basis of the calculated t-test, olive oil, NaNO3 and level of inoculums were the most significant variables affecting the biosurfactant production. Their interactions are shown in Fig.7 (a, b, c) which had shown increase of biosurfactant production with the increase of olive oil, NaNO3, and level of inoculum, indicating an indirect

relationship between these three factors for high yield of biosurfactant. In the further studies, these three variables would be subjected to Response Surface Methodology to conform the optimized production media for the high yield of the biosurfactant by Streptomyces coelicoflavus.

Fig 6: Elucidation of fermentation conditions affecting the Biosurfactant production by Streptomyces coelicoflavus

(a) (b) (c)

Fig 7: Interaction between (a) Olive oil and NaNO3 (b) Olive oil and level of inoculum (c) NaNO3 and level of inoculum

concentrations 4. Conclusion The present study focused primarily on improved biosurfactant production by Streptomyces coelicoflavus NRBC 15399T as a function of various salt compositions and levels of ingredients in production medium. The Plackett-Burman design was found to be very effective in selecting and optimizing the medium components in a manageable number of experimental trials with an increase in biosurfactant production. Moreover, the optimized culture medium obtained in this experiment will be useful for further study with large scale fermentation in a fermenter for the efficient biosurfactant production. 5. Acknowledgment The author expresses her gratitude to Prof. G. Girija Sankar and Prof. T. Prabhakar (A.U. College of Pharmaceutical Sciences) for their immense help and valuable advises for this research. 6. References 1. Chandrasekaran E, BeMiller JN, Song-Chiau DL.

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